Sequence and chiral selectivity of DNA-drug interactions revealed by force spectroscopy

ABSTRACT

Methods of quantifying the efficiency of a drug molecule for its targeted receptor, using a differential binding force to quantify the efficiency of a drug molecule to its targeted receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Stage under 35 U.S.C. §371of International Patent Application No. PCT/US2015/052355, filed Sep.25, 2015, which claims priority to U.S. Provisional Application No.62/055,897 filed Sep. 26, 2014, the disclosures of which are hereinincorporated in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grants No.ECCS-1028328 and ECCS-1508845 both of which were awarded by the UnitedStates National Science Foundation. The United States government hascertain rights in the invention.

BACKGROUND

Field of the Disclosure

This disclosure generally relates to methods of quantifying theefficiency of a drug molecule for its targeted receptor. Moreparticularly, this disclosure is drawn to methods of using adifferential binding force to quantify the efficiency of a drug moleculeto its targeted receptor. Further, this disclosure relates to a methodof quantifying: enantomeric selectivity of drugs for a target; theselectivity of a series of drugs for a target; and the selectivity of aDNA sequence for binding to a receptor/target or ligand.

Background of the Technology

The interactions between small molecules, such as ions and drugmolecules, and nucleic acids are widely encountered in biologicalfunctions and drug development. Among the most important aspects incharacterizing these systems are the sequence selectivity of the DNA andthe conformational selectivity of the drug molecules. Various techniqueshave been applied to study drug-DNA systems, including nuclear magneticresonance (NMR), second harmonic generation, fluorescence, circulardichroism UV melting,^([9]) X-ray,^([10]) atomic force microscopy(AFM),^([11,12]) and optical tweezers. However, none of these techniquescan adequately, and directly measure the binding strength of suchinteractions with a sufficiently high force resolution, so thatdifferent DNA-drug interactions can be distinguished. Therefore, amethod to quantify the efficiency of a drug molecule to its targetedreceptor; a method of quantitatively measuring DNA sequence selectivityto a ligand or a receptor; and the enantomeric selectivity of a moleculefor its receptor/target would be well received in the field.

BRIEF SUMMARY OF THE DISCLOSED EMBODIMENTS

In order to address such needs, the disclosure provides in someembodiments described herein: a method of quantifying the efficiency ofa drug molecule to a receptor by measuring a differential binding force.The method comprises: (a) conjugating a magnetic particle to a ligand toform a magnetic particle-ligand conjugate; (b) adding said conjugate toa receptor, wherein said receptor is immobilized on a surface, andforming a ligand-receptor complex; (c) measuring a first magnetizationof said complex; (d) subjecting said complex to a gradually increasingforce, wherein said force is increased in increments, and measuring themagnetization at each incremental force value; (e) measuring thedissociation force (F1) of the complex, (f) reforming the complex; (g)adding a drug molecule to said complex to form a second complex; oralternatively, adding the drug molecule to the receptor prior to step f,then adding the ligand to form the second complex; (h) measuring thedissociation force (F2) of said second complex by repeating steps d ande; and (i) subtracting F1 from F2 to quantify the differential bindingforce of said drug molecule to said ligand-receptor complex. In someembodiments of the method, in step e, said dissociation of the complexoccurs when consecutive magnetization values decrease by a maximum value(wherein the absolute slope of the magnetization-force curve reaches amaximum value). In some embodiments the force range may be 0.1 to about900 pN. In some other embodiments of the method of quantifying theefficiency of a drug molecule to a receptor by measuring a differentialbinding force the ligand is selected from a group comprising nucleicacids and proteins, in other embodiments the receptor is selected from agroup comprising nucleic acids and proteins. In further embodiments, thedrug molecule is selected from the group comprising synthetic organicmolecules and molecules extracted from natural products, and in stillfurther embodiments the drug molecule is label-free. In otherembodiments of the method of quantifying the efficiency of a drugmolecule to a receptor by measuring a differential binding force, thedrug molecule is a racemic mixture, in further embodiments the drugmolecule is an enantiomer.

In another embodiment a method of determining enantiomeric selectivityof a drug for a target is disclosed; the method comprises: (a)conjugating a magnetic particle to a ligand to form a magneticparticle-ligand conjugate; (b) adding said conjugate to a receptor,wherein said receptor is immobilized on a surface, and forming aligand-receptor complex; (c) measuring a first magnetization of saidcomplex; (d) subjecting said complex to a gradually increasing force,wherein said force is increased in increments, and measuring themagnetization at each incremental force value (wherein the forceincrement depends on the mass of the magnetic particles and thecentrifugal speed, and comprises a range in some embodiments of 0.1-10pN, and in further embodiments 1-5 pN); (e) measuring the dissociationforce (F1) of the complex, (f) reforming the complex; (g) adding a firstenantiomer to said complex to form a second complex; or alternatively,adding the first enantiomer to the receptor prior to step f, then addingthe ligand to form the second complex; (h) measuring the dissociationforce (F2) of said second complex by repeating steps d and e; (i)subtracting F1 from F2 to quantify the differential binding force ofsaid first enantiomer to said ligand-receptor complex; (j) adding asecond enantiomer to said complex to form a second-enantiomer complexcomprising the second enantiomer; or alternatively, adding the secondenantiomer to the receptor prior to step f, then adding the ligand toform the second-enantiomer complex comprising the second enantiomer; (k)measuring the dissociation force (F2′) of said second-enantiomer complexcomprising the second enantiomer by repeating steps d and e; (l)subtracting F1 from F2′ to quantify the differential binding force ofsaid second enantiomer to said ligand-receptor complex; (m) subtractingsaid differential binding force of said second enantiomer from saiddifferential binding force of said first enantiomer; and determiningwhich enantiomer is most tightly bound to said receptor. In furtherembodiments of the method, at step (e), and step (c) the dissociation ofthe complex occurs when consecutive magnetization values decrease by amaximum value. In another embodiment, of the method of determiningenantiomeric selectivity of a drug for a target, the ligand is selectedfrom a group comprising nucleic acids and proteins, in anotherembodiment the receptor is selected from a group comprising nucleicacids and proteins, in a further embodiment the drug molecule isselected from the group comprising synthetic organic molecules andmolecules extracted from natural products, and in a still furtherembodiment the drug molecule is label-free.

In another embodiment, a method of determining the selectivity of anucleic acid sequence for a drug molecule is disclosed; the methodcomprises: (a) conjugating a magnetic particle to a ligand to form amagnetic particle-ligand conjugate; (b) adding said conjugate to a firstreceptor wherein said first receptor comprises a first nucleic acidsequence, and wherein said first receptor is immobilized on a surfaceand forming a ligand-first-receptor complex; (c) measuring a firstmagnetization of said ligand-first-receptor complex; (d) subjecting saidligand-first-receptor complex to a gradually increasing force, whereinsaid force is increased in increments, and measuring the magnetizationat each incremental force value; (e) measuring the dissociation force(F1) of the ligand-first-receptor complex, (f) reforming theligand-first-receptor complex; (g) adding a drug molecule to saidcomplex to form a second ligand-first-receptor complex; oralternatively, adding the drug molecule to the receptor prior to step f,then adding the ligand to form the second ligand-first-receptor complex;(h) measuring the dissociation force (F2) of said second complex byrepeating steps d and e; (i) subtracting F1 from F2 to quantify thedifferential binding force of said drug molecule to saidligand-first-receptor complex; (j) adding said conjugate to a secondreceptor, wherein said second receptor comprises a second nucleic acidsequence that differs from said first receptor by at least one nucleicacid, and wherein said receptor is immobilized on a surface and forminga ligand-second-receptor complex; (k) measuring a first magnetization ofsaid ligand-second-receptor complex; (l) subjecting saidligand-second-receptor complex to a gradually increasing force, whereinsaid force is increased in increments, and measuring the magnetizationat each incremental force value; (m) measuring the dissociation force(F1′) of the ligand-second-receptor complex, (n) reforming theligand-second-receptor complex; (o) adding a drug molecule to saidligand-second-receptor complex to form a second ligand-second-receptorcomplex; or alternatively, adding the drug molecule to the receptorprior to step (f), then adding the ligand to form the secondligand-second-receptor complex; (p) measuring the dissociation force(F2′) of said second ligand-second-receptor complex by repeating steps dand e; (q) subtracting F1′ from F2′ to quantify the differential bindingforce of said drug molecule to said ligand-second-receptor complex; (r)comparing said differential binding force of said drug molecule to saidligand-second-receptor complex and the differential binding force ofsaid drug molecule to said ligand-first-receptor complex; and (s)determining from step r the selectivity of said nucleic acid sequence ofsaid receptors for said drug molecule. In another embodiment, wherein instep (e), and step (c) the dissociation of the complex occurs whenconsecutive magnetization values decrease by a maximum value. In afurther embodiment, the ligand is selected from a group comprisingnucleic acids and proteins, in a still further embodiment, said firstand second receptors are selected from a group comprising nucleic acidsand proteins; in another embodiment the drug molecule is selected fromthe group comprising synthetic organic molecules and molecules extractedfrom natural products; and in a further embodiment the drug molecule islabel-free.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

It should be understood at that although an illustrative implementationof one or more embodiments are provided below, the disclosed systemsand/or methods may be implemented using any number of techniques,whether currently known or in existence. The disclosure should in no waybe limited to the illustrative implementations, drawings, and techniquesbelow, including the exemplary designs and implementations illustratedand described herein, but may be modified within the scope of theappended claims along with their full scope of equivalents.

The following discussion is directed to various exemplary embodiments ofthe invention. However, the embodiments disclosed should not beinterpreted, or otherwise used, as limiting the scope of the disclosure,including the claims. In addition, one skilled in the art willunderstand that the following description has broad application, and thediscussion of any embodiment is meant only to be exemplary of thatembodiment, and that the scope of this disclosure, including the claims,is not limited to that embodiment.

In the following discussion and in the claims, the terms “including” and“comprising” are used in an open-ended fashion, and thus should beinterpreted to mean “including, but not limited to . . . .” Also, theterm “couple” or “couples” is intended to mean either an indirect ordirect connection. Thus, if a first device couples to a second device,that connection may be through a direct engagement between the twodevices, or through an indirect connection via other intermediatedevices and connections. As used herein, the term “about,” when used inconjunction with a percentage or other numerical amount, means plus orminus 10% of that percentage or other numerical amount. For example, theterm “about 80%,” would encompass 80% plus or minus 8%.

Force-induced remnant magnetization spectroscopy (FIRMS) technique usesexternal mechanical forces to distinguish noncovelant bonds includingDNA binding. The force resolution has been shown to reached 1.8 pN, andis sufficient to resolve DNA duplexes with one basepair difference (seefor example U.S. Pat. No. 8,802,057 incorporated herein in its entiretyby reference).^([16])

The external force may include shaking force, centrifugal force, or anacoustic radiation force. The FIRMS technique is based on the fact thatdissociated magnetic particles that label biological molecules have nonet magnetic signal because of their randomized magnetic dipoles.Therefore, as each bond dissociates and the magnetic particle is nolonger bound it is effectively measured because there is a decrease inthe overall magnetic signal.

Herein, embodiments of a method to quantify the efficiency (of binding)of a drug molecule to its targeted receptor are disclosed. Further amethod of quantitatively measuring a DNA sequences' selectivity to aligand or a target/receptor is also disclosed; further, a method ofquantifying the enantomeric selectivity of a molecule for itsreceptor/target is disclosed, thereby using differential binding forceto precisely characterize the mechanical effect of a molecular speciesbinding to a target. Therefore, the high-resolution binding forcesmeasured by the FIRMS technique disclosed herein distinguishes thebinding mode/behavior among drug molecules of different chirality; DNAof various sequences; and drug molecules of differing targetselectivity, all of which are critical in drug design and mechanisticstudies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: depicts a schematic of an embodiment of a method describedherein: wherein one strand of the DNA duplex is immobilized on asurface, while the other is labeled with a magnetic particle. Thebinding forces of the DNA duplex are measured in the absence andpresence of the drug molecule, denoted as F₁ and F₂, respectively. Thedifferential binding force, F₂−F₁, characterizes the influence of thedrug-DNA binding on the stability of the DNA (the binding forces areobtained as described herein);

FIG. 2: graphically depicts the measurements of the T-Hg-T binding forceand shows the comparison with the differential binding forces of T-Hg-Twith A-T and C-G pairs;

FIG. 3: depicts DNA sequence specificity (as measured by an embodimentof the method herein) for daunomycin binding. a) shows specific bindingof daunomycin with a DNA duplex, DNA₁. b) shows nonbinding betweendaunomycin with a second DNA, DNA₂;

FIG. 4: depicts sequence specificity and chiral selectivity of DNAintercalating with tetrahydropalmatine (THP), wherein a) DNA₁selectively binds with d-THP; and b) DNA₂ selectively binds with l-THP;

FIG. 5: depicts a mapping of differential binding forces for two DNAsequences with intercalating drug molecules, daunomycin and (d, l)-THP.Wherein a cross signifies zero differential binding force; and a circlesignifies positive differential binding force, with circular areaindicating the relative amplitude;

FIG. 6: depicts the differential binding forces of two groove-bindingdrugs, netropsin and berenil. Their binding mode is different from theintercalation interaction of daunomycin and THP;

FIG. 7: depicts a graph showing the magnetic signals (a) red (leftplot), before bond dissociation, and (b) blue (right plot), after bonddissociation; and

FIG. 8: depicts the molecular structures of daunomycin and d, l-THP.

EXAMPLES

Some embodiments of the method of using a differential binding force toquantify the efficiency of a drug molecule to a receptor comprises aprocess wherein one strand of the DNA duplex is immobilized on asurface, while the other is labeled with a magnetic particle. Thebinding forces of the DNA duplex are measured in the absence andpresence of the drug molecule, denoted as F₁ and F₂, respectively. Thedifferential binding force, F₂−F₁, characterizes the influence of thedrug-DNA binding on the stability of the DNA. The binding forces areobtained using the FIRMS technique, depicted in FIG. 1.

Example 1 Characterizing DNA Sequence Specificity for a Ligand

An embodiment of the method herein described is used to quantify thebinding modes of the T-Hg-T system (T, thymine), in which Hg refers toHg²⁺. This system has been studied by NMR as well as other spectroscopictechniques. It is therefore known that Hg specifically intercalates intoa DNA duplex at the T-T mismatching pair. Based on the previous results,the binding of T-Hg-T is weaker than that of C-G but stronger than A-T.FIG. 2 graphically depicts the measurements of the T-Hg-T binding forceand comparison with the differential binding forces of A-T and C-Gpairs, as measured by the method described herein and depicted in FIG.1.

Experimental Details:

Magnetic particles M280 were used to label one strand of the DNAduplexes as previously characterized. After initial magnetization by apermanent magnet, the magnetic signal of the particles were detected byan atomic magnetometer, with a sensitivity of 200 fT/(Hz)^(1/2). Samplewells (4×2×1 mm³) with a biotin-coated bottom surface were used toimmobilize the other strand of the DNA duplexes. Mechanical forces wereapplied using a centrifuge (Eppendorf 5417R).

The sample well, with dimensions of 4×2×1 mm³ (L×W×D), was coated withbiotin on the bottom surface. An aqueous solution of 0.625 mg/mLstreptavidin was loaded into the sample well and incubated for 1 hr.Then the sample well was rinsed three times with a buffer solution asdescribed below. 8 □L of 10 □M biotinylated target DNA strand wastransferred onto the streptavidin-decorated surface and incubated for 1hr. After rinsing the surface, 8 □L of 10 □M biotinylated probing DNAstrand was in contact with the target DNA-modified surface overnight.The formed DNA duplex was rinsed with buffer solution. Subsequently, 8□L of 1% bovine serum album (BSA) was introduced into the sample welland incubated for 1 hr before the addition of the streptavidin-coatedmagnetic particles (Invitrogen, M280). The particles were pre-washedthree times with the buffer solution. After incubation for 2 hrs, thephysically absorbed magnetic particles were removed from the surface byapplying centrifuge with the speed of 1000 rpm for 5 min. The sample wasthen magnetized for 2 min using a permanent magnet (˜0.5 T). Further,the intercalators of interest, including Hg²⁺, daunomycin, and d- andl-THP, were introduced into the surface-immobilized magnetic particlesimmersed under the buffer solution and kept for 1 hr. Forces withvarying amplitudes were applied on the samples by gradually increasingthe speed of the centrifuge. The centrifuge time for each speed was 5min.

Magnetization measurements were performed using scanning magneticimaging with an atomic magnetometer with a sensitivity of about 200fT/√Hz. For the T-Hg-T system, 10 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH=7.6)containing 0.5 M NaNO₃ and 0.05% Tween-20 was used. The intercalation ofdaunomycin into DNA duplexes was performed using the TE buffer (50 mMTris, 138 mM NaCl, 2.7 mM KCl, 1 mM EDTA, and 0.05% Tween-20, pH=8.0).The interactions between THP and DNA duplexes were investigated withBPES buffer (6 mM Na₂HPO₄, 2 mM NaH₂PO₄, 185 mM NaCl, 1 mM EDTA, and0.05% Tween-20, pH=7.0).

Magnetic signals before and after bond dissociation: The magneticparticles in the sample well were magnetized only once by a permanentmagnet, wherein a decrease in the remnant magnetic signal represents thedissociation of the noncovalent bonds (J. Phys. Chem. B 117, 7554-7558(2013). The magnetic detection was obtained using a scanning magneticimaging method (Angew. Chem. Int. Ed. 48, 5679-5682 (2009)). FIG. 7,thus shows the embodiments of magnetic field profiles before and afterthe dissociation of the DNA duplex with an additional C-G pairing in theT-Hg-T experiments. The results correspond to the data points at 58 and61 pN on the blue trace in FIG. 2 (far right trace).

The sudden decrease in the magnetic signal from 3800 rpm to 3900 rpmindicates the dissociation of the DNA duplex. Given the buoyant mass ofthe particles being 4.6×10⁻¹⁵ kg and the radius of the centrifuge of 8cm, the binding forces were calculated by the method herein described(and depicted in FIG. 1) to be to be 58 and 62 pN respectively,according to the equation: F=m□²r (J. Phys. Chem. B 117, 7554-7558(2013)).

Therefore, the force resolution calculated by an embodiment of themethod herein described is approximately 2 pN. The DNA sequence used forHg binding was:

-   -   5′-CCC GGG TTT CCC-3′    -   3′-GGG CCC AAT GGG-5′,        which contains a T-T pair as underlined (SEQ. 1 and SEQ. 2        respectively). The binding force of the DNA duplex was        determined to be 40 pN, calculated from the buoyant mass of the        magnetic particles, the centrifugal speed at which the        dissociation occurred, and the radius of the centrifuge.

Upon binding with Hg, the binding force increased to 54 pN, representinga 14 pN increase. For comparison, the T-T pair was replaced in theduplex with A-T and C-G respectively. Their binding forces were measuredto be 51 and 60 pN, respectively. The differential binding forces arethereby 11 pN for A-T and 20 pN for C-G, in this particular DNAplatform. The results are consistent with AFM results, which gave 9±3 pNfor A-T binding and 20±3 pN for C-G pairing. Typical magnetic signalsbefore and after DNA duplex dissociation are depicted in FIG. 7.Comparing the differential binding forces as calculated herein, leads tothe following binding order: A-T<T-Hg-T<C-G, and is consistent with theorder of melting points in the literature. Therefore, the resultsvalidate the application of FIRMS measurement of differential bindingforces for characterizing the binding between small molecules and DNAduplexes. (Qiongzheng Hu, and Shoujun Xu, Sequence and ChiralSelectivity of DNA-Drug Interactions Revealed by Force Spectroscopy,Angewandte Chemie International Edition, Vol. 53, 51, pg 14135-14138,Dec. 15, 2014).

Example 2 Elucidation and Quantification of Sequence Selectivity

Two DNA molecules were herein tested for their binding selectivity bythe methods herein disclosed (chemical structures are shown in FIG. 8).The drug molecule tested was daunomycin, a commonly used anticancerdrug, which preferentially binds to specific triplex sequences in DNAduplexes.

DNA Intercalation is widely considered as its binding mode, althoughminor groove binding has also been discussed, however, the specificityhas previously not been quantified by binding strength. In order tomeasure the binding forces of daunomycin into different DNA duplexes,the target DNA strand used was 5′-CCC AAT CGA CCC-3′ (SEQ. 3), whereinthe probe DNA was a 12 bp complementary DNA strand, 5′-GGG TCG ATTGGG-3′ (SEQ. 4). The duplex is represented as DNA₁. Based on theprevious reports, daunomycin may have specifically bound to the CGAsegment. As a control experiment the differential binding force ofdaunomycin with a different DNA duplex, DNA₂, with sequence 5′-CCC GGGTTT CCC-3′ and its complementary strand was measured. DNA₂, however doesnot contain a CGA segment.

The results of the binding experiments are shown in FIG. 3. Uponintercalating with daunomycin, the binding force of DNA₁ increased from45 pN to 57 pN. In contrast, no significant force difference wasobserved in the control experiment with DNA₂: the binding force was 51pN for the DNA and 52 pN for DNA₂-daunomycin complex. The results areconsistent with the literature in that daunomycin specifically targetthe CGA sequence. The differential binding force of 12 pN shows that thecontribution of daunomycin intercalation to the thermal stability issimilar to that of the A-T pairing, but substantially smaller than thatof C-G pairing. Thus the drug molecule is specific for DNA₁ as clearlymeasured by the differential binding energy of 12 pN.

Example Three Elucidation & Quantification of Enantomeric Selectivity

In a further embodiment the method herein described was applied toanother drug molecule: tetrahydropalmatine (THP). THP is a naturalalkaloid racemate extracted from Rhizoma Corydalis. Racemic d/l THP isincluded in the active compounds that result in the antitumor effect ofCorydalis.

Although it has been reported that racemic THP shows enantioselectivebinding to DNA using gas chromatography, the features for the d-THP andl-THP were largely unresolved. In addition, the investigation oninteractions between THP and DNA duplex is rare. It remained previouslyunknown whether the binding force of the DNA duplex could be increasedafter incubation of the alkaloid with DNA duplexes, which is valuablefor understanding the antitumor activity of the drug molecule.

The binding of THP with the two DNA sequences used in the previousdaunomycin experiment was performed. Both d- and l-THP were studied witheach DNA sequence. The results are shown in FIG. 4. For DNA₁ (FIG. 4a ),the binding of l-THP led to the binding force of the duplex increasingfrom 44 pN to 54 pN, producing a differential binding force of 10 pN.This value shows that effective binding took place between DNA₁ andd-THP, with a binding strength similar to the A-T pairing. Conversely,DNA₁ incubated with l-THP did not yield a binding force increase. Thecomplex had a binding force of 43 pN, thus no significant bindingoccurred between DNA₁ and l-THP.

The behaviors of both chiral molecules bound with DNA₂ were however inopposite fashion. In FIG. 4b , the binding force of DNA₂-d-THP complexwas 51 pN, which is essentially the same as that of the DNA alone.However, DNA₂-l-THP complex increased the binding force to 66 pN. Thedifferential binding force of 15 pN is in between the binding of A-T andC-G pairing.

The differential binding forces of the various drug-DNA systemsdescribed herein, and measured by embodiments of the method of measuringthe differential binding forces are further summarized in FIG. 5. Thismap clearly shows the mutual selectivity between DNA and drug molecules.For instance, DNA₁ binds with both daunomycin and d-THP, with the formerbeing slightly stronger than the latter, whereas it does notsignificantly bind with l-THP. In some complicated cases, it may becommon to have many similar drug structures and multiple potential DNAtargets, such a map of differential binding force will therefore providea clear way of representing binding selectivities. The high-resolutionFIRMS method described herein therefore accurately determines thedifferential binding force and therefore the enantomeric and sequenceselectivities of a drug molecule interacting a nucleotide sequence.

Example Four Quantification of Groove Binding Between DNA and Drugs

The method of differential binding force can also be used to quantify adifferent binding mode of DNA-drug interactions. We chose twogroove-binding drugs, netropsin and berenil. The groove-bindinginteraction is different from the intercalation interaction ofdaunomycin and THP. Shown in FIG. 6 are the binding forces of DNA alone,DNA with netropsin, and DNA with berenil, with values of 59, 63, and 73pN, respectively. The DNA sequence was 5′-CGCGAAAAACGCG-3′ (SEQ. 5). Thebinding forces yield differential binding force of 4 pN for netropsinand 14 pN for berenil. The results clearly show that berenil bindsstronger to the target DNA duplex.

The method of using differential binding force is not limited todrug-DNA systems. It may be of equal value in general ligand-receptorsystems involving drug molecules. The binding of the drug molecule willalter the effectiveness of the ligand-receptor recognition. Furthermore,the differential binding force does not have to be positive; a negativevalue will indicate reduced binding specificity between the ligand andreceptor molecules. Possible examples may be inhibitors, which aredesigned to block the receptors from their corresponding ligands. Drugresistance studies may also be probed, where single point mutations innucleotides will result in changes of binding forces for a probemolecule, or SAR may be probed for a series of drugs and a target in ahigh throughput fashion.

Therefore, embodiments of the method herein provided measuredifferential binding forces by using high-resolution FIRMS, and may beutilized in (but not limited to) drug screening and other applicationsinvolving noncovalent molecular binding. A further benefit of themethods described herein is that the small molecules under study are notlabeled, this is of value in practical applications because of thedifficulty of labeling small molecules and the consequent interferencesof the labeling groups in binding studies.

All references cited herein are incorporated in their entireties byreference. Further, while exemplary embodiments of the invention havebeen shown and described, modifications thereof can be made by oneskilled in the art without departing from the spirit and teachings ofthose embodiments. The embodiments described herein are exemplary only,and are not intended to be limiting. Many variations and modificationsof the disclosed embodiments are possible and are within the scope ofthe claimed invention. Where numerical ranges or limitations areexpressly stated, such express ranges or limitations should beunderstood to include iterative ranges or limitations of like magnitudefalling within the expressly stated ranges or limitations (e.g., fromabout 1 to about 10 includes, 2, 3, 4, etc.; greater than 0.10 includes0.11, 0.12, 0.13, etc.). For example, whenever a numerical range with alower limit, R_(l), and an upper limit, R_(u), is disclosed, any numberfalling within the range is specifically disclosed. In particular, thefollowing numbers within the range are specifically disclosed:R=R_(l)+k* (R_(u)−R_(l)), wherein k is a variable ranging from 1 percentto 100 percent with a 1 percent increment, i.e., k is 1 percent, 2percent, 3 percent, 4 percent, 5 percent, . . . 50 percent, 51 percent,52 percent, . . . , 95 percent, 96 percent, 97 percent, 98 percent, 99percent, or 100 percent. Moreover, any numerical range defined by two Rnumbers as defined in the above is also specifically disclosed. Use ofthe term “optionally” with respect to any element of a claim is intendedto mean that the subject element is required, or alternatively, is notrequired. Both alternatives are intended to be within the scope of theclaim. Use of broader terms such as comprises, includes, having, etc.should be understood to provide support for narrower terms such asconsisting of, consisting essentially of, comprised substantially of,etc.

Accordingly, the scope of protection is not limited by the descriptionset out above but is only limited by the claims which follow, that scopeincluding all equivalents of the subject matter of the claims. Each andevery claim is incorporated into the specification as an embodiment ofthe present invention. Thus, the claims are a further description andare an addition to the embodiments of the present invention. Thedisclosures of all patents, patent applications, and publications citedherein are hereby incorporated by reference, to the extent that theyprovide exemplary, procedural or other details supplementary to thoseset forth herein.

The invention claimed is:
 1. A method of quantifying the efficiency of adrug molecule to a receptor by measuring a differential binding force,the method comprising: a) conjugating a magnetic particle to a ligand toform a magnetic particle-ligand conjugate; b) adding said conjugate to areceptor, wherein said receptor is immobilized on a surface and forminga ligand-receptor complex; c) measuring a first magnetization of saidcomplex; d) subjecting said complex to a gradually increasing force,wherein said force is increased in increments, and measuring themagnetization at each incremental force value; e) determining thedissociation force (F1) of the complex; f) reforming the complex; g)adding a drug molecule to said complex to form a second complex; oralternatively, adding the drug molecule to the receptor prior to step f,then adding the ligand to form the second complex; h) measuring thedissociation force (F2) of said second complex by repeating steps d ande; i) subtracting F1 from F2 to quantify the differential binding forceof said drug molecule to said ligand-receptor complex.
 2. The method ofclaim 1, wherein in step e, said dissociation of the complex occurs whenconsecutive magnetization values decrease by a maximum value.
 3. Themethod of claim 1, wherein in said ligand is selected from a groupcomprising nucleic acids and proteins.
 4. The method of claim 1, whereinsaid receptor is selected from a group comprising nucleic acids andproteins.
 5. The method of claim 1, wherein said drug molecule isselected from the group comprising synthetic organic molecules andmolecules extracted from natural products.
 6. The method of claim 1,wherein said drug molecule is label-free.
 7. The method of claim 1,wherein said drug molecule is a racemic mixture.
 8. The method of claim7, wherein said drug molecule is an enantiomer.
 9. A method ofdetermining enantiomeric selectivity of a drug for a target; the methodcomprising: a) conjugating a magnetic particle to a ligand to form amagnetic particle-ligand conjugate; b) adding said conjugate to areceptor, wherein said receptor is immobilized on a surface and forminga ligand-receptor complex; c) measuring a first magnetization of saidcomplex; d) subjecting said complex to a gradually increasing force,wherein said force is increased in increments, and measuring themagnetization at each incremental force value; e) determining thedissociation force (F1) of the complex; f) reforming the complex; g)adding a first enantiomer to said complex to form a second complex; oralternatively, adding the first enantiomer to the receptor prior to stepf, then adding the ligand to form the second complex; h) measuring thedissociation force (F2) of said second complex by repeating steps d ande; i) subtracting F1 from F2 to quantify the differential binding forceof said first enantiomer to said ligand-receptor complex; j) adding asecond enantiomer to said complex to form a second-enantiomer complexcomprising the second enantiomer; or alternatively, adding the secondenantiomer to the receptor prior to step f, then adding the ligand toform the second-enantiomer complex comprising the second enantiomer; k)measuring the dissociation force (F2′) of said second-enantiomer complexcomprising the second enantiomer by repeating steps d and e; l)subtracting F1 from F2′ to quantify the differential binding force ofsaid second enantiomer to said ligand-receptor complex; m) subtractingsaid differential binding force of said second enantiomer from saiddifferential binding force of said first enantiomer; and determiningwhich enantiomer is most tightly bound to said receptor.
 10. The methodof claim 9, wherein in step e, and step c said dissociation of thecomplex occurs when consecutive magnetization values decrease by amaximum value.
 11. The method of claim 9, wherein in said ligand isselected from a group comprising nucleic acids and proteins.
 12. Themethod of claim 9, wherein said receptor is selected from a groupcomprising nucleic acids and proteins.
 13. The method of claim 9,wherein said drug molecule is selected from the group comprisingsynthetic organic molecules and molecules extracted from naturalproducts.
 14. The method of claim 9, wherein said drug molecule islabel-free.
 15. A method of determining the selectivity of a nucleicacid sequence for a drug molecule, the method comprising: a) conjugatinga magnetic particle to a ligand to form a magnetic particle-ligandconjugate; b) adding said conjugate to a first receptor wherein saidfirst receptor comprises a first nucleic acid sequence, and wherein saidfirst receptor is immobilized on a surface and forming aligand-first-receptor complex; c) measuring a first magnetization ofsaid ligand-first-receptor complex; d) subjecting saidligand-first-receptor complex to a gradually increasing force, whereinsaid force is increased in increments, and measuring the magnetizationat each incremental force value; e) determining the dissociation force(F1) of the ligand-first-receptor complex; f) reforming theligand-first-receptor complex; g) adding a drug molecule to said complexto form a second ligand-first-receptor complex; or alternatively, addingthe drug molecule to the receptor prior to step f, then adding theligand to form the second ligand-first-receptor complex; h) measuringthe dissociation force (F2) of said second complex by repeating steps dand e; i) subtracting F1 from F2 to quantify the differential bindingforce of said drug molecule to said ligand-first-receptor complex; j)adding said conjugate to a second receptor, wherein said second receptorcomprises a second nucleic acid sequence that differs from said firstreceptor by at least one nucleic acid, and wherein said receptor isimmobilized on a surface and forming a ligand-second-receptor complex;k) measuring a first magnetization of said ligand-second-receptorcomplex; l) subjecting said ligand-second-receptor complex to agradually increasing force, wherein said force is increased inincrements, and measuring the magnetization at each incremental forcevalue; m) measuring the dissociation force (F1′) of theligand-second-receptor complex, n) reforming the ligand-second-receptorcomplex; o) adding a drug molecule to said ligand-second-receptorcomplex to form a second ligand-second-receptor complex; oralternatively, adding the drug molecule to the receptor prior to step f,then adding the ligand to form the second ligand-second-receptorcomplex; p) measuring the dissociation force (F2′) of said secondligand-second-receptor complex by repeating steps d and e; q)subtracting F1′ from F2′ to quantify the differential binding force ofsaid drug molecule to said ligand-second-receptor complex; r) comparingsaid differential binding force of said drug molecule to saidligand-second-receptor complex and the differential binding force ofsaid drug molecule to said ligand-first-receptor complex; and s)determining from step r the selectivity of said nucleic acid sequence ofsaid receptors for said drug molecule.
 16. The method of claim 14,wherein in step e, and step c said dissociation of the complex occurswhen consecutive magnetization values decrease by a maximum value. 17.The method of claim 14, wherein in said ligand is selected from a groupcomprising nucleic acids and proteins.
 18. The method of claim 14,wherein said first and second receptors are selected from a groupcomprising nucleic acids and proteins.
 19. The method of claim 14,wherein said drug molecule is selected from the group comprisingsynthetic organic molecules and molecules extracted from naturalproducts.
 20. The method of claim 14, wherein said drug molecule islabel-free.